Effects of 3-nitrooxypropanol on methane emission,digestion, and energy and nitrogen balance of lactating dairy cows

The objective was to measure effects of 3-nitrooxypropanol (3NP) on methane production of lactating dairy cows and any associated changes in digestion and energy and nitrogen metabolism. Six Holstein-Friesian dairy cows in mid-lactation were fed twice daily a total mixed ration with maize silage as the primary forage source. Cows received 1 of 3 treatments using an experimental design based on two 3 × 3 Latin squares with 5-wk periods. Treatments were a control placebo or 500 or 2,500 mg/d of 3NP delivered directly into the rumen, via the rumen fistula, in equal doses before each feeding. Measurements of methane production and energy and nitrogen balance were obtained during wk 5 of each period using respiration calorimeters and digestion trials. Measurements of rumen pH (48 h) and postprandial volatile fatty acid and ammonia concentrations were made at the end of wk 4. Daily methane production was reduced by 3NP, but the effects were not dose dependent (reductions of 6.6 and 9.8% for 500 and 2,500 mg/d, respectively). Dosing 3NP had a transitory inhibitory effect on methane production, which may have been due to the product leaving the rumen in liquid outflow or through absorption or metabolism. Changes in rumen concentrations of volatile fatty acids indicated that the pattern of rumen fermentation was affected by both doses of the product, with a decrease in acetate:propionate ratio observed, but that acetate production was inhibited by the higher dose. Dry matter, organic matter, acid detergent fiber, N, and energy digestibility were reduced at the higher dose of the product. The decrease in digestible energy supply was not completely countered by the decrease in methane excretion such that metabolizable energy supply, metabolizable energy concentration of the diet, and net energy balance (milk plus tissue energy) were reduced by the highest dose of 3NP. Similarly, the decrease in nitrogen digestibility at the higher dose of the product was associated with a decrease in body nitrogen balance that was not observed for the lower dose. Milk yield and milk fat concentration and fatty acid composition were not affected but milk protein concentration was greater for the higher dose of 3NP. Twice-daily rumen dosing of 3NP reduced methane production by lactating dairy cows, but the dose of 2,500 mg/d reduced rumen acetate concentration, diet digestibility, and energy supply. Further research is warranted to determine the optimal dose and delivery method of the product. Key words: 3-nitrooxypropanol, methane, digestion, rumen, dairy cow

Splanchnic metabolism of nitrogenous compounds and urinary nitrogen excretion in steers fed alfalfa under conditions of increased absorption of ammonia and L-arginine supply across the portal-drained viscera

Effects of increased ammonia and/or arginine absorption across the portal-drained viscera (PDV) on net splanchnic (PDV and liver) metabolism of nitrogenous compounds and urinary N excretion were investigated in six cathetenzed Hereford x Angus steers (501 +/- 1 kg BW) fed a 75% alfalfa:25% (as-fed basis) corn-soybean meal diet (0.523 MJ of ME/[kg BW0.15.d]) every 2 h without (27.0 g of N/kg of dietary DM) and with 20 g of urea/kg of dietary DM (35.7 g of N/kg of dietary DM) in a split-plot design. Net splanchnic flux measurements were obtained immediately before beginning and ending a 72-h mesenteric vein infusion of L-arginine (15 mmol/h). For 3 d before and during arginine infusion, daily urine voided was measured and analyzed for N composition. Feeding urea increased PDV absorption (P < 0.01) and hepatic removal (P < 0.01) of ammonia N, accounting for 80% of increased hepatic urea N output (P < 0.01). Numerical increases in net hepatic removal of AA N could account for the remaining portion of increased hepatic urea N output. Arginine infusion increased hepatic arginine removal (P < 0.01) and hepatic urea N output (P < 0.03) and switched hepatic ornithine flux from net uptake to net output (P < 0.01), but numerical changes in net hepatic removal of ammonia and AA N could not account fully for the increase in hepatic urea N output. Increases in urine N excretion equaled quantities of N fed as urea or infused as arginine. Estimated salivary urea N excretion was not changed by either treatment. Urea cycle regulation occurs via a complex interaction of mechanisms and requires N sources other than ammonia, but the effect of increased ammonia absorption on hepatic catabolism of individual AA in the present study was not significant.

An integrative model of amino acid metabolism in the liver of the lactating dairy cow

The objective of this work was to construct a dynamic model of hepatic amino acid metabolism in the lactating dairy cow that could be parameterized using net flow data from in vivo experiments. The model considers 22 amino acids, ammonia, urea, and 13 energetic metabolites, and was parameterized using a steady-state balance model and two in vivo, net flow experiments conducted with mid-lactation dairy cows. Extracellular flows were derived directly from the observed data. An optimization routine was used to derive nine intracellular flows. The resulting dynamic model was found to be stable across a range of inputs suggesting that it can be perturbed and applied to other physiological states. Although nitrogen was generally in balance, leucine was in slight deficit compared to predicted needs for export protein synthesis, suggesting that an alternative source of leucine (e.g. peptides) was utilized. Simulations of varying glucagon concentrations indicated that an additional 5 mol/d of glucose could be synthesized at the reference substrate concentrations and blood flows. The increased glucose production was supported by increased removal from blood of lactate, glutamate, aspartate, alanine, asparagine, and glutamine. As glucose Output increased, ketone body and acetate release increased while CO2 release declined. The pattern of amino acids appearing in hepatic vein blood was affected by changes in amino acid concentration in portal vein blood, portal blood flow rate and glucagon concentration, with methionine and phenylalanine being the most affected of essential amino acids. Experimental evidence is insufficient to determine whether essential amino acids are affected by varying gluconeogenic demands. (C) 2004 Published by Elsevier Ltd.

Effect of prolonged intravenous glucose and essential amino acid infusion on nitrogen balance, muscle protein degradation and ubiquitin-conjugating enzyme gene expression in calves

Background: Intravenous infusions of glucose and amino acids increase both nitrogen balance and muscle accretion. We hypothesised that co-infusion of glucose ( to stimulate insulin) and essential amino acids (EAA) would act additively to improve nitrogen balance by decreasing muscle protein degradation in association with alterations in muscle expression of components of the ubiquitin-proteasome proteolytic pathway. Methods: We examined the effect of a 5 day intravenous infusions of saline, glucose, EAA and glucose + EAA, on urinary nitrogen excretion and muscle protein degradation. We carried out the study in 6 restrained calves since ruminants offer the advantage that muscle protein degradation can be assessed by excretion of 3 methyl-histidine and multiple muscle biopsies can be taken from the same animal. On the final day of infusion blood samples were taken for hormone and metabolite measurement and muscle biopsies for expression of ubiquitin, the 14-kDa E2 ubiquitin conjugating enzyme, and proteasome sub-units C2 and C8. Results: On day 5 of glucose infusion, plasma glucose, insulin and IGF-1 concentrations were increased while urea nitrogen excretion and myofibrillar protein degradation was decreased. Co-infusion of glucose + EAA prevented the loss of urinary nitrogen observed with EAA infusions alone and enhanced the increase in plasma IGF-1 concentration but there was no synergistic effect of glucose + EAA on the decrease in myofibrillar protein degradation. Muscle mRNA expression of the ubiquitin conjugating enzyme, 14-kDa E2 and proteasome sub-unit C2 were significantly decreased, after glucose but not amino acid infusions, and there was no further response to the combined infusions of glucose + EAA. Conclusion: Prolonged glucose infusion decreases myofibrillar protein degradation, prevents the excretion of infused EAA, and acts additively with EAA to increase plasma IGF-1 and improve net nitrogen balance. There was no evidence of synergistic effects between glucose + EAA infusion on muscle protein degradation or expression of components of the ubiquitin-proteasome proteolytic pathway.

Metabolism of Rhizobium bacteroids

Nitrogen fixation within legume nodules results from a complex metabolic exchange between bacteria of the family Rhizobiaciae and the plant host. Carbon is supplied to the differentiated bacterial cells, termed bacteroids, in the form of dicarboxylic acids to fuel nitrogen fixation. In exchange, fixed nitrogen is transferred to the plant. Both the bacteroid and the plant-derived peribacteroid membrane tightly regulate the exchange of metabolites. In the bacteroid oxidation of dicarboxylic acids via the TCA cycle occurs in an oxygen-limited environment. This restricts the TCA cycle at key points, such as the 2-oxoglutarate dehydrogenase complex, and requires that inputs of carbon and reductant are balanced with outputs from the TCA cycle. This may be achieved by metabolism through accessory pathways that can remove intermediates, reductant, or ATP from the cycle. These include synthesis of the carbon polymers PHB and glycogen and bypass pathways such as the recently identified 2-oxoglutarate decarboxylase reaction in soybean bacteroids. Recent labeling data have shown that bacteroids synthesize and secrete amino acids, which has led to controversy over the role of amino acids in nodule metabolism. Here we review bacteroid carbon metabolism in detail, evaluate the labeling studies that relate to amino acid metabolism by bacteroids, and place the work in context with the genome sequences of Mesorhizobium loti and Sinorhizobium meliloti. We also consider a wider range of metabolic pathways that are probably of great importance to rhizobia in the rhizosphere, during nodule initiation, infection thread development, and bacteroid development.

Strategies for optimizing nitrogen use by ruminants

The efficiency of N utilization in ruminants is typically low (around 25%) and highly variable (10% to 40%) compared with the higher efficiency of other production animals. The low efficiency has implications for the production performance and environment. Many efforts have been devoted to improving the efficiency of N utilization in ruminants, and while major improvements in our understanding of N requirements and metabolism have been achieved, the overall efficiency remains low. In general, maximal efficiency of N utilization will only occur at the expense of some losses in production performance. However, optimal production and N utilization may be achieved through the understanding of the key mechanisms involved in the control of N metabolism. Key factors in the rumen include the efficiency of N capture in the rumen (grams of bacterial N per grams of rumen available N) and the modification of protein degradation. Traditionally, protein degradation has been modulated by modifying the feed (physical and chemical treatments). Modifying the rumen microflora involved in peptide degradation and amino acid deamination offers an alternative approach that needs to be addressed. Current evidence indicates that in typical feeding conditions there is limited net recycling of N into the rumen (blood urea-N uptake minus ammonia-N absorption), but understanding the factors controlling urea transport across the rumen wall may reverse the balance to take advantage of the recycling capabilities of ruminants. Finally, there is considerable metabolism of amino acids (AA) in the portal-drained viscera (PDV) and liver. However, most of this process occurs through the uptake of AA from the arterial blood and not during the ‘absorptive’ process. Therefore, AA are available to the peripheral circulation and to the mammary gland before being used by PDV and the liver. In these conditions, the mammary gland plays a key role in determining the efficiency of N utilization because the PDV and liver will use AA in excess of those required by the mammary gland. Protein synthesis in the mammary gland appears to be tightly regulated by local and systemic signals. The understanding of factors regulating AA supply and absorption in the mammary gland, and the synthesis of milk protein should allow the formulation of diets that increase total AA uptake by the mammary gland and thus reduce AA utilization by PDV and the liver. A better understanding of these key processes should allow the development of strategies to improve the efficiency of N utilization in ruminants.

Energy and protein interactions and their effect on nitrogen excretion in dairy cows

The principal driver of nitrogen (N) losses from the body including excretion and secretion in milk is N intake. However, other covariates may also play a role in modifying the partitioning of N. This study tests the hypothesis that N partitioning in dairy cows is affected by energy and protein interactions. A database containing 470 dairy cow observations was collated from calorimetry experiments. The data include N and energy parameters of the diet and N utilization by the animal. Univariate and multivariate meta-analyses that considered both within and between study effects were conducted to generate prediction equations based on N intake alone or with an energy component. The univariate models showed that there was a strong positive linear relationships between N intake and N excretion in faeces, urine and milk. The slopes were 0.28 faeces N, 0.38 urine N and 0.20 milk N. Multivariate model analysis did not improve the fit. Metabolizable energy intake had a significant positive effect on the amount of milk N in proportion to faeces and urine N, which is also supported by other studies. Another measure of energy considered as a covariate to N intake was diet quality or metabolizability (the concentration of metabolizable energy relative to gross energy of the diet). Diet quality also had a positive linear relationship with the proportion of milk N relative to N excreted in faeces and urine. Metabolizability had the largest effect on faeces N due to lower protein digestibility of low quality diets. Urine N was also affected by diet quality and the magnitude of the effect was higher than for milk N. This research shows that including a measure of diet quality as a covariate with N intake in a model of N execration can enhance our understanding of the effects of diet composition on N losses from dairy cows. The new prediction equations developed in this study could be used to monitor N losses from dairy systems.

Novel European free-living, non-diazotrophic Bradyrhizobium isolates from contrasting soils that lack nodulation and nitrogen fixation genes - a genome comparison

The slow-growing genus Bradyrhizobium is biologically important in soils, with different representatives found to perform a range of biochemical functions including photosynthesis, induction of root nodules and symbiotic nitrogen fixation and denitrification. Consequently, the role of the genus in soil ecology and biogeochemical transformations is of agricultural and environmental significance. Some isolates of Bradyrhizobium have been shown to be non-symbiotic and do not possess the ability to form nodules. Here we present the genome and gene annotations of two such free-living Bradyrhizobium isolates, named G22 and BF49, from soils with differing long-term management regimes (grassland and bare fallow respectively) in addition to carbon metabolism analysis. These Bradyrhizobium isolates are the first to be isolated and sequenced from European soil and are the first free-living Bradyrhizobium isolates, lacking both nodulation and nitrogen fixation genes, to have their genomes sequenced and assembled from cultured samples. The G22 and BF49 genomes are distinctly different with respect to size and number of genes; the grassland isolate also contains a plasmid. There are also a number of functional differences between these isolates and other published genomes, suggesting that this ubiquitous genus is extremely heterogeneous and has roles within the community not including symbiotic nitrogen fixation.